Developing strategies and a toolbox for metabolic engineering of thermophiles for ethanol production

Completed

Renewable Energy Sources(Bio-Energy, Production of transport biofuels (incl. Production from wastes))
Renewable Energy Sources(Bio-Energy, Production of other biomass-derived fuels (incl. Production from wastes))

Basic and strategic applied research

BIOLOGICAL AND AGRICULTURAL SCIENCES (Biological Sciences)

Not Cross-cutting

Prof DJ Leak
Biology and Biochemistry
University of Bath

Research Grant

BBSRC

01 February 2007

31 May 2010

40 months

£338,088

Pharmaceuticals and Biotechnology

South West

Prof DJ Leak, Biology and Biochemistry, University of Bath

Objectives not supplied

Future bioethanol production will need to use cellulose and hemicellulose from biomass (eg short rotation crops and plant waste) in a multi-step 'biorefinery'. Ideally, this will involve continuous production (and ethanol removal eg at high temperatures) with organisms which grow on pentoses, hexoses and complex polysaccarides and tolerate processed biomass derived toxins. We will explore two strategies to convert any genetically amenable glycolytic thermophile into an ethanol producer. The first is to create a thermophilic pyruvate decarboxylase. The enzyme from Zymomonas mobilis, although moderately thermostable once folded, does not fold correctly in Geobacillus spp above 50oC, probably because the affinity between the unfolded enzyme and TDP-Mg2+ cofactors is too low. Using selection based on growth, we will use directed evolution methods including error prone PCR and oligonucleotide shuffling (based on sequences encoding TDP-Mg2+ binding sites from thermophiles), to incrementally improve the thermophilicity of PDC. The resulting PDC gene may then be combined with that for a thermophilic alcohol dehydrogenase (ADH) to create a metabolic engineering 'cassette' that could be applied in other thermophiles. The second strategy exploits the observation that pyruvate dehydrogenase, together with elements of the pyruvate formate lyase pathway have a redox balance and metabolic outcome identical to that of PDC-ADH. As all of these functions are present in thermophiles it should be possible to create a novel homoethanol fermentation pathway based on PDH, CoA dependent acetaldehyde dehydrogenase and ADH. Approaches for anaerobic expression of PDH will be explored, together with the possibility of converting non/poorly-fermenting strains into ethanol producers. In the latter context we will test G. denitrificans K1041 under conditions of low nitrate availability. Finally, we intend to apply these tools to a newly isolated 'process optimised' thermophile.

07/10/13